Bottom Line:
Missense mutations were found for all genes while truncations were selected for all except one.We estimated that, on average, these lines carry one mutation every 89 kb, Ler population providing a total of more than five million induced mutations.Analysis of TILLer collection demonstrates its usefulness for large scale TILLING reverse genetics in another reference genetic background of A. thaliana.

Background: Arabidopsis thaliana is the main model species for plant molecular genetics studies and world-wide efforts are devoted to identify the function of all its genes. To this end, reverse genetics by TILLING (Targeting Induced Local Lesions IN Genomes) in a permanent collection of chemically induced mutants is providing a unique resource in Columbia genetic background. In this work, we aim to extend TILLING resources available in A. thaliana by developing a new population of ethyl methanesulphonate (EMS) induced mutants in the second commonest reference strain. In addition, we pursue to saturate the number of EMS induced mutations that can be tolerated by viable and fertile plants.

Results: By mutagenizing with different EMS concentrations we have developed a permanent collection of 3712 M2/M3 independent mutant lines in the reference strain Landsberg erecta (Ler) of A. thaliana. This population has been named as the Arabidopsis TILLer collection. The frequency of mutations per line was maximized by using M1 plants with low but sufficient seed fertility. Application of TILLING to search for mutants in 14 genes identified 21 to 46 mutations per gene, which correspond to a total of 450 mutations. Missense mutations were found for all genes while truncations were selected for all except one. We estimated that, on average, these lines carry one mutation every 89 kb, Ler population providing a total of more than five million induced mutations. It is estimated that TILLer collection shows a two to three fold higher EMS mutation density per individual than previously reported A. thaliana population.

Conclusions: Analysis of TILLer collection demonstrates its usefulness for large scale TILLING reverse genetics in another reference genetic background of A. thaliana. Comparisons with TILLING populations in other organisms indicate that this new A. thaliana collection carries the highest chemically induced mutation density per individual known in diploid species.

Mentions:
To obtain a new population of chemically induced mutant lines useful for reverse genetic studies in Arabidopsis thaliana, seeds of the Landsberg erecta (Ler) glabrous1-1 genotype were mutagenized with EMS at concentrations of 20 to 50 mM (Figure 1). The effects of EMS and the efficiency of the mutagenesis treatment were estimated by quantifying three parameters on M1 plants: seed germination, frequency of albino chimeras and fertility (see Methods). Germination of M1 seeds was negatively correlated with EMS dose (r = -0.93; p = 0.008), while the frequency of M1 albino chimeras increased with concentration (r = 0.99; p = 0.001) (Figure 1A and 1B). Seed fertility of M1 plants and the degree of M2 embryo lethality was quantified by estimating the proportion of fully or nearly sterile fruits (classes As and Aa) and the proportion of semi or normal fertile fruits (classes B and C). As shown in Figure 1C, the total frequency of class A fruits increased linearly with EMS concentration, whereas the frequency of fertile fruits rapidly decreased. To maximize the frequency of mutations per individual, only M1 plants from treatments showing a frequency of fertile fruits smaller than 35% but larger than 2% were individually harvested. A total of 3712 M2 families were grown to isolate individual M2 DNA and to harvest their M3 offspring seeds. To ensure independence of the mutations present in this population, a single M2 plant was harvested from each M1 plant. In agreement with the high proportion of embryo lethality, all M2 families segregated for easily visible morphological mutations (data not shown). Fifty six percent of M2 lines were derived from 25 mM EMS mutagenesis, and on average, EMS treatments used to obtain the collection show less than 25% fertile fruits (Table 1). The DNA of M2 plants and the M3 seeds of the 3712 lines were stored (see Methods) providing a permanent population of mutant lines for TILLING analysis in Ler genetic background. This population has been named as the Arabidopsis TILLer collection.

Mentions:
To obtain a new population of chemically induced mutant lines useful for reverse genetic studies in Arabidopsis thaliana, seeds of the Landsberg erecta (Ler) glabrous1-1 genotype were mutagenized with EMS at concentrations of 20 to 50 mM (Figure 1). The effects of EMS and the efficiency of the mutagenesis treatment were estimated by quantifying three parameters on M1 plants: seed germination, frequency of albino chimeras and fertility (see Methods). Germination of M1 seeds was negatively correlated with EMS dose (r = -0.93; p = 0.008), while the frequency of M1 albino chimeras increased with concentration (r = 0.99; p = 0.001) (Figure 1A and 1B). Seed fertility of M1 plants and the degree of M2 embryo lethality was quantified by estimating the proportion of fully or nearly sterile fruits (classes As and Aa) and the proportion of semi or normal fertile fruits (classes B and C). As shown in Figure 1C, the total frequency of class A fruits increased linearly with EMS concentration, whereas the frequency of fertile fruits rapidly decreased. To maximize the frequency of mutations per individual, only M1 plants from treatments showing a frequency of fertile fruits smaller than 35% but larger than 2% were individually harvested. A total of 3712 M2 families were grown to isolate individual M2 DNA and to harvest their M3 offspring seeds. To ensure independence of the mutations present in this population, a single M2 plant was harvested from each M1 plant. In agreement with the high proportion of embryo lethality, all M2 families segregated for easily visible morphological mutations (data not shown). Fifty six percent of M2 lines were derived from 25 mM EMS mutagenesis, and on average, EMS treatments used to obtain the collection show less than 25% fertile fruits (Table 1). The DNA of M2 plants and the M3 seeds of the 3712 lines were stored (see Methods) providing a permanent population of mutant lines for TILLING analysis in Ler genetic background. This population has been named as the Arabidopsis TILLer collection.

Bottom Line:
Missense mutations were found for all genes while truncations were selected for all except one.We estimated that, on average, these lines carry one mutation every 89 kb, Ler population providing a total of more than five million induced mutations.Analysis of TILLer collection demonstrates its usefulness for large scale TILLING reverse genetics in another reference genetic background of A. thaliana.

Background: Arabidopsis thaliana is the main model species for plant molecular genetics studies and world-wide efforts are devoted to identify the function of all its genes. To this end, reverse genetics by TILLING (Targeting Induced Local Lesions IN Genomes) in a permanent collection of chemically induced mutants is providing a unique resource in Columbia genetic background. In this work, we aim to extend TILLING resources available in A. thaliana by developing a new population of ethyl methanesulphonate (EMS) induced mutants in the second commonest reference strain. In addition, we pursue to saturate the number of EMS induced mutations that can be tolerated by viable and fertile plants.

Results: By mutagenizing with different EMS concentrations we have developed a permanent collection of 3712 M2/M3 independent mutant lines in the reference strain Landsberg erecta (Ler) of A. thaliana. This population has been named as the Arabidopsis TILLer collection. The frequency of mutations per line was maximized by using M1 plants with low but sufficient seed fertility. Application of TILLING to search for mutants in 14 genes identified 21 to 46 mutations per gene, which correspond to a total of 450 mutations. Missense mutations were found for all genes while truncations were selected for all except one. We estimated that, on average, these lines carry one mutation every 89 kb, Ler population providing a total of more than five million induced mutations. It is estimated that TILLer collection shows a two to three fold higher EMS mutation density per individual than previously reported A. thaliana population.

Conclusions: Analysis of TILLer collection demonstrates its usefulness for large scale TILLING reverse genetics in another reference genetic background of A. thaliana. Comparisons with TILLING populations in other organisms indicate that this new A. thaliana collection carries the highest chemically induced mutation density per individual known in diploid species.